Efficiency of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli

Biochemistry. 1994 Aug 16;33(32):9552-60. doi: 10.1021/bi00198a022.

Abstract

We have measured the rates and efficiencies of DNA unwinding (the number of ATP molecules hydrolyzed per DNA base pair unwound) catalyzed by the RecBC,RecBCD-K177Q (a site-directed mutant in the putative ATP-binding site in the RecD subunit), and RecBCD enzymes from Escherichia coli. The DNA unwinding rate was measured with a coupled assay in which unwound DNA is degraded by the combined action of the RecJ enzyme and exonuclease I. The rates of DNA unwinding by the RecBC and RecBCD-K177Q enzymes are reduced by about 4-fold compared to the case of the RecBCD enzyme. The efficiency of ATP hydrolysis was determined in two ways. First, it was calculated from the ratio of the ATP hydrolysis rate to the rate of DNA unwinding. In the second method, ATP hydrolysis was measured under conditions where all of the DNA substrate becomes completely unwound. The efficiency is the ratio of the total amount of ATP hydrolyzed to the amount of DNA substrate present in the reaction. The average efficiencies measured kinetically and by the complete unwinding experiment are as follows: 2.30 and 1.74 ATP/base pair (RecBCD enzyme); 1.44 and 1.28 (RecBC); and 1.20 and 1.07 (RecBCD-K177Q). The RecBC and RecBCD-K177Q enzymes are therefore able to couple ATP hydrolysis to DNA unwinding at least as efficiently as the RecBCD holoenzyme. The lower ATP per base pair ratios found for RecBC and RecBCD-K177Q indicate that the RecD subunit hydrolyzes ATP during DNA unwinding by the RecBCD enzyme.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Bacterial Proteins / metabolism
  • DNA Helicases / metabolism*
  • DNA, Bacterial / metabolism*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism*
  • Hydrolysis
  • Models, Biological
  • Nucleic Acid Conformation
  • Protein Binding

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Adenosine Triphosphate
  • Exodeoxyribonucleases
  • RecJ protein, E coli
  • recJ protein, Bacteria
  • exodeoxyribonuclease I
  • Exodeoxyribonuclease V
  • exodeoxyribonuclease V, E coli
  • DNA Helicases